Epidemiological studies have repeatedly shown that reproductive processes in pregnant women are adversely affected by exposure to cigarette smoke. The potential reproductive targets of smoke during pregnancy include the ovaries, oviducts, uterus, placenta, umbilical cord, and embryo/fetus. In vitro methods for studying the effects of smoke and its individual components have been developed and applied to each of these reproductive targets. In vitro assays have been useful in determining the biological processes that are affected in the reproductive organs and in identifying the cellular and molecular targets of smoke in each organ. In vitro methods have also been used to study the mechanism of action of smoke constituents, such as nicotine, on specific processes in reproductive organs and to screen smoke solutions to identify the molecules that affect reproduction. In general, data collected in vitro have confirmed, extended, and helped explain what has been learned from epidemiological studies. This review summarizes some of the in vitro assays that have been used to study cigarette smoke's effect on the nonpregnant and pregnant female reproductive tract and spotlights examples of their applications.